Isolation and characterization of biosurfactant/biopolymer producing spore forming bacteria from oil contaminated sites and oil field of Oman

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1 Available online at APCBEE Procedia 5 (2013 ) ICESD 2013: January 19-20, Dubai, UAE Isolation and characterization of biosurfactant/biopolymer producing spore forming bacteria from oil contaminated sites and oil field of Oman S.N. Al-Bahry a, A.E. Elshafie a, Y.M. Al-Wahaibi b, A.S. Al-Bemani b, S. J. Joshi a, A. Al-Lawati a a Department of Biology, College of Science, Sultan Qaboos University, Sultanate of Oman b Petroleum & Chemical Engineering Department, College of Engineering, Sultan Qaboos University, Sultanate of Oman Abstract Biosurfactants and biopolymers are key players for microbial enhanced oil recovery (MEOR) mechanisms. These biomolecules can be produced In-situ or Ex-situ and can be utilised for enhancing oil recovery. In this study, several aerobic spore forming bacteria were isolated from diverse habitats for ex-situ MEOR. The selected 16 isolates were identified by 16s rrna sequencing and screened for better biosurfactant and biopolymer production using different production medium at shake flask level. One Bacillus subtilis strain W19, was found to be a promising biosurfactant producer and it reduced ST and IFT from 72 to 27mN/m and from 46 to 3.3mN/m respectively in less than 20 hours The Published Authors. by Published Elsevier by B.V. Elsevier Selection B.V. Open and/or access peer under review CC BY-NC-ND under responsibility license. of Asia-Pacific Selection Chemical, and Biological peer review & under Environmental responsibility Engineering of Asia-Pacific Society Chemical, Biological & Environmental Engineering Society Keywords: Biosurfactant; biopolymer; Microbial-enhanced-oil-recovery; spore-formers; Bacillus subtilis 1. Introduction Various chemical or physical (for e.g., thermal) enhanced or improved oil recovery (EOR or IOR) technologies are used worldwide to treat declining oil reservoirs. Amongst those techniques, considerable interest has been generated towards using microbiological techniques for enhancing oil recovery known as Corresponding author. Tel.: ; fax: address: snbahry@squ.edu.om The Authors. Published by Elsevier B.V. Open access under CC BY-NC-ND license. Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi: /j.apcbee

2 S.N. Al-Bahry et al. / APCBEE Procedia 5 ( 2013 ) microbial enhanced oil recovery (MEOR). MEOR have several advantages over chemical methods, including environmental compatibility [1]. Biosurfactant, biopolymers, and biomass are amongst the key players in MEOR. It has been reported that several spore-forming bacilli are producing potent biosurfactant, which reduces IFT between water and oil to ultra-low IFT. Biosurfactants aid in recovering oil by reducing IFT between oil and water, whereas, biopolymer increases the viscosity of formation water and thus aids in EOR. Several spore-forming bacteria were isolated in this investigation from local garages and oil field, and screened them for better biosurfactant production. Selected isolates were further screened using different carbohydrates based minimal media for better biosurfactant producer. 2. Experimental procedures Samples were collected from different garages and petrol stations in Oman (from Al-Hayl, Al-Maabila, Al- Gala, Al-Wadi Al-Kabeer) and from Wafra oil field, Al-Bahja area. The samples were placed in sterile polyethylene bags or sterile flasks and stored at 4 Samples were boiled at 100 for 45 minutes. After cooling, the samples were serially diluted and plated on agar containing media plates using Autoplate 4000, Spiral Biotech, USA. The plates were incubated for 72 hours at 40, 50 and 70, and checked for wellisolated colonies. The biosurfactant production was screened by oil spreading technique [2]. For production of biosurfactant, microbes were inoculated in 50ml of Mineral Salt Media (MSM) [3]. The highest biosurfactant activity was indicated by the largest diameter of the clear zone and the best isolates were selected for further study to measure the activity of the biosurfactant in minimal media. After primary screening of biosurfactant producing isolates, the positive isolates were studied by different morphological and biochemical tests and identified by 16s rrna sequencing. Bacterial DNA was extracted by the 'Ultra Clean TM ' Microbial soil DNA [4]. A standard PCR processes was used and the primers sets which used were: - AGAGTTTGATCCTGGCTCAG- -GGTTACCTTGTTACGACTT- Identification of microbes was done by gene sequencer at Macrogen Sequencing Service, South Korea; and the microbes were identified. The selected isolates were studied for their potential of biosurfactant, biopolymer and biomass production in 9 different reported production media (Table 1) for Bacilli. The samples were analyzed for biosurfactant production (Surface Tension and Interfacial Tension), biopolymer (viscosity), and microbial growth (OD 660 ) every 24 hours for 72 hours. All measurements were made on cell-free broth after centrifugation (11,292 g for 20 min), and were analyzed at room temperatures. Production of biosurfactant was analyzed by measurement of ST and IFT using pendant drop method using the Drop Shape Analyzer, DSA 100 (KRUSS, Germany). For biopolymer production, 1 ml sample were analyzed by viscosity measurement using quartz viscometer (F5 technology, Germany). The experiments were performed in duplicate and the results reported are the mean of three independent experiments. 3. Results and Discussion Total of 72 spore-forming bacterial isolates were isolated from garage samples and oil well samples, out of which 16 were selected for further studies, based on the results of oil-spreading technique. Those 16 isolates showed bigger zone of oil-spreading as compared to rest of the isolates (Fig. 1), and were given the code names - B2, B5, B6, B7, B13, B17, B23, B25, B26, B28, B29, W7, W16, W19, W28 and W32. The 16 Bacilli isolates were identified using 16s rrna sequencing and 15 of them were identified as Bacillus licheniformis and one as B. subtilis. The sequences were deposited in National Centre of Biotechnology

3 244 S.N. Al-Bahry et al. / APCBEE Procedia 5 ( 2013 ) Information (NCBI) GenBank and the isolates were given Accession Numbers GU GU Table 1. Production media composition for biosurfactant production Production Medium Composition (g/l) 1 [5] 2 [6] 3a [7] 3b 4 [8] 5 [9] 6 [10] 7 [11] 8 [12] Cane Molasses Date Molasses Sucrose Glucose NH 4 NO Na- Glutamate NaNO K 2 HPO KH 2 PO Na 2 HPO MgSO 4.7H 2 O FeSO 4.7H 2 O MnSO 4.4H 2 O KCl CaCl Na-EDTA H 3 PO 4 (85.4%) ml ml CuSO Yeast Extract NaCl (NH 4 ) 2 SO Trace elements* - 10 ml ml 10 ml - 0.5ml The 16 selected isolates were studied for their potential of biosurfactant, biopolymer and biomass production in 9 different production media. For initial screening, amongst 16 Bacilli isolates, 9 different media were used with 2% (w/v) carbohydrate as carbon source. The samples were analysed for biosurfactant production (ST and IFT), biopolymer (viscosity), acid production (ph change) and microbial growth (OD 660 ) every 24 hours for 72 hours. Amongst all 16 Bacilli, B. subtilis W19 showed highest reduction in ST and IFT in production media 5, 7 and 8. The results are shown in figures 2 (A-D). 24 h. Whereas, ST (<25mN/m) and IFT (~5mN/m) were reduced prominently in media 5, 7 and 8. There was no significant variation of viscosity in all 9 media, but highest viscosities (~1.5mPa.s) were observed in media 3a (cane molasses medium) and medium 5 (Fig 2C). No acid production was observed as the ph became alkaline in all media after 72h, except in media 1 & 4 where it dropped to 6.0 after 72 h. Significantly biomass (OD 660

4 S.N. Al-Bahry et al. / APCBEE Procedia 5 ( 2013 ) Fig. 1. Clear zone diameters by selected 16 Bacilli isolates using oil-spreading technique (A) (B) (C) (D) Fig. 2. Biosurfactant (A, B), biopolymer (C), and biomass (D) production by B. subtilis W19

5 246 S.N. Al-Bahry et al. / APCBEE Procedia 5 ( 2013 ) Previous researchers reported that aerobic microorganisms present in the well salinity are predominant by gram positive, facultative and spore forming rod bacteria [13]. Kowalewski et al. [14] reported the induced effect of bacteria on increase of wettability of rock and reduction of IFT on oil recovery. Youssef et al., [2] examined in their study B. subtilis and B. licheniformis and other species related to them their ability to have MEOR characteristics. In the present study, out of 72 spore formers, only 16 Bacillus species were capable to growth under aerobic and anaerobic conditions, able to survive up to 70ºC, which is an oil well condition and capable to grow at high saline conditions similar to the oil well salinity ( ppm). B. subtilis W19 was found to be a promising biosurfactant producer and it reduced ST and IFT from 72 to 27mN/m and from 46 to 3.3mN/m respectively in less than 20 hours. It should be studied further for enhanced oil recovery experiments. Acknowledgements Authors acknowledge the Sultan Qaboos University, Oman (His Majesty Project: SR/SCI/BIOL/08/01) and the Petroleum Development of Oman (CR/SCI/BIOL/07/02) for the their full financial support. References [1] Al-Sulaimani H, Joshi S, Al-Wahaibi Y, Elshafie A, Al-Bahry SA, Al-Bemani A. Microbial biotechnology for enhancing oil recovery, prospects and developments. Review paper. Biotechnol Bioinfo Bioeng J 2011;1: [2] Youssef NH, Duncan KE, McInerney MJ. Importance of 3-Hydroxy fatty acid composition of lipopeptides for biosurfactant activity. Appl Env Microbiol 2005;71: [3] Banat I M. The isolation of a thermophilic biosurfactant producing Bacillus sp. Biotechnol Lett 1993;15: [4] Yamane K, Maki H, Nakayama T, Nakajima T, Nomura N, Uchiyama H, Kitaoka M. Diversity and similarity of microbial communities in petroleum crude oils produced in Asia. Biosci Biotechnol Biochem 2008;72: [5] Joshi, S, Yadav S, Desai A. Application of response surface methodology to evaluate the optimum medium components for the enhanced production of lichenysin by B. licheniformis R2. Biochem Eng J 2008;41: [6] Joshi S, Yadav S, Nerurkar A, Desai A. Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51. J Microbiol Biotechnol 2007;17: [7] Joshi S, Bharucha C, Jha S, Yadav S, Nerurkar A, Desai A. Biosurfactant production using substrates from renewable-resources, molasses and whey under thermophilic conditions Biores technol 2008;99: [8] Landy MG, Warren H, Rosenman SB, Colio LG. Bacillomycin, an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc Soc Exp Biol Med 1948;67: [9] Jenny K, Kappeli O, Fiechter A. Biosurfactant from Bacillus licheniformis: structural analysis and characterization. Appl Microbiol Biotechnol 1991;36:5-13. [10] Youssef N, Simpson DR, Duncan KE, McInerney MJ, Flomsbee M, Fincher T, Knapp RM. In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. Appl Env Microbiol 2007;73: [11] Cooper DG, Macdonald CR, Duff SJ, Kosaric N. Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl Env Microbiol 1981;42: [12] Mukherjee S, Das P, Sen RK. Rapid quantification of a microbial surfactant by a simple turbidometric method. J Microbiol Methods 2009;76: [13] Illlas RM, Wei OS, Idris AK, Rahman WA. Isolation and characterization of halotolerant aerobic bacteria from oil reservoir. J Teknologi 2001;35:1-10. [14] Kowalewski E, Rueslatten I, Steen KH, Bodtker G, Torsaeter O. Microbial improved oil recovery-bacterial induced wettability and interfacial tension effects on oil production. J Petrol Sci Eng 2006;52: